Construction Of The Small Ligand Three-Hybrid System

Proliferation of the leukemia K562 cells could be inhibited by matrine, but the molecular mechanism is unknown, especially the target interacted with matrine. So we plan to construct the small ligand three-hybrid system to detect the target protein interacted with matrine in vivo.The objects of present study included three parts: 1. Amplification of combination domain of glucocorticoid receptor gene in K562 cells with RT-PCR. 2. Construction, transformation and expression of the binding domain expression plasmid pGBKT7-GR. 3.Preparation of the human erythroleukemia MATCHMAKER cDNA library and study of the influence of the small ligand on yeast cells growthing and mating.Fragments of glucocorticoid receptor binding domain were amplified,constructed to recombine pGBKT7-GR vector and confirmed by sequencing. Recombinant pGBKT7-GR vector were transformed to AH109 cells. Toxicity and self-activation of DNA-BD fusion proteins were detected and had no significant effect on the yeast hybrid system, while the...